Method for optimizing therapeutic efficacy of nemorubicin

ABSTRACT

The present invention relates to products and methods for characterizing cancer patients in order to predict therapeutic benefit with a drug metabolized by CYP3A, especially nemorubicin.

FIELD OF THE INVENTION

The present invention pertains to a new approach to anticancertreatments. More specifically, the invention relates to methods fordetermining a patient individualized dosage of a drug primarilymetabolized by cytocrome P4503A isoenzyme, especially nemorubicin, inorder to decrease toxicity and increase efficacy of the chemotherapeutictreatments.

DESCRIPTION OF THE INVENTION

The present invention relates to the field of cancer treatment and, moreparticularly, it relates to a method for optimizing the treatment ofcancer with chemotherapy by measuring cytocrome P4503A isoenzyme (CYP3A)levels in patients undergoing chemotherapeutic treatment.

It has recently been found that nemorubicin is metabolized primarily byCYP3A4.

The cytocrome P450 (CYP) enzymes constitute a large superfamily ofhaem-containing proteins that play a central role in the metabolism of awide variety of endogenous compounds and foreign chemicals, includingdrugs (Nelson et al, Pharmacogenetics, 1996). In mammals, the main drugmetabolizing families of CYP (CYP1, CYP2, CYP3) are primarily expressedin the liver, although specific isoforms are present in someextrahepatic tissues (de Waziers et al, J. Pharmacol. Exp. Ther. 1990).CYP3A4, the most abundantly expressed CYP enzyme in adult human liver,may account for the oxidative metabolism of more than 60% of allclinically used drugs, including anticancer agents such ascyclophosphamide, ifosfamide, paclitaxel, vinblastine andepipodophyllotoxins (Chang et al Cancer Res. 1993; Kivisto et al., Br.J. Clin Pharmacol. 1995; Shimada et al., J. Pharmacol. Exp. Ther. 1994).

It was reported that there was a great interindividual variation in theCYP expression (Shimada et al., J. Pharmacol. Exp. Ther. 1994).

Moreover, CYP3A enzymes are expressed at different levels in humantumors (de Waziers et al, J. Pharmacol. Exp. Ther. 1990; Murray et al.,J. Pathol. 1995), and can be inhibited or induced by a number of drugs(Waxman, Arch Biochem. Biophys. 1999). Thus, the expression of CYP3A mayprofoundly affect the activity and/or the host toxicity of antitumoragents, which are substrates of these enzymes. Moreover, clinicallyapplicable techniques capable of predicting CYP3A4-levels in humans areavailable (Rivory et al. Clin. Cancer Res. 2000).

Nemorubicin is a doxorubicin derivative currently undergoing clinicalevaluation. Previous studies suggest that nemorubicin undergoes hepaticbiotransformation into a more cytotoxic metabolite(s). These metaboliteshave been identified and their antitumor activity and toxicity have beentested (Geroni et al., Proc. Am. Assoc. Cancer Res., 1997). Inexperimental tumor models, all tested metabolites of nemorubicinresulted as active as the parent compound. As regards potency, one ofthe identified metabolites presented higher potency in respect tonemorubicin, being its maximum tolerated dose more than five times lowerthan that of the parent compound.

More recently, the metabolic pathway of nemorubicin has beeninvestigated.

The following EXPERIMENTAL PART illustrates, for example, the role ofCYP3A in the metabolic pathway of nemorubicin.

Experimental Part

Materials and Methods

Antibody Studies

Human liver microsomes were preincubated at 25° C. for 5 min. with andwithout a monoclonal antibody anti-CYP3A4/5 (MAB-3A4 Genetest) in Tris0.3M (pH 7.4) before adding nemorubicin (20 μM and NADPH (0.5 mM). After10 min at 37° C., the amount of nemorubicin metabolites was evaluated bya HPLC system.

Metabolic Potential of Microsomes Obtained from Cells Expressing SingleHuman CYP 450 Isoenzymes

Microsomes were obtained from cell cultures overexpressing CYP3A4,CYP3A5, CYP1A2, CYP2E1, CYP2D61, CYP2C91 and CYP2C8. Microsomes (50 pmolCYP/ml) were incubated with nemomubicin (20 μM) and NADPH (0.5 mM) in0.3M Tris (pH 7.4) at 37° C. for 20 min. Nemorubicin metabolism wasquantified by HPLC method.

HPLC Analysis

High-performance liqid chromatographic (HPLC) system consisted of aWaters Model 510 isocratic pump equipped with autosampler. Detection wasperformed by a Perkin Elmer fluorescence spectrofotometer LS-5 set at479 and 552 nm excitation and emission wavelength, respectively.Detector was connected to a Shimadzu C-R3A integrator. Thecromatographic separation was performed on a Waters Simmetry C8 reversephase column. The mobile phase was 10 mM KH₂P₄/Methanol/CH₃CN(45:30:25). The flow rate was 1.5 ml/min. Standard curves offluorescence versus drug concentration for nemorubicin and metaboliteswere used to calculate drug concentration in the samples.

Correlation Between CYP3A Expression and Nemorubicin Metabolism in HumanMicrosomes from Different Patients

Nemorubicin was incubated with liver microsomes from 9 differentpatients. All human liver microsome samples were tested for theexpression of CYP3A by erythromycin-demethylase test (Watkins et al. J.Clin. Invest. 1993). The amount of nemorubicin metabolites was evaluatedby a HPLC system and correlated to the expression of CYP3A.

Results

The metabolism of nemorubicin correlates with the levels of CYP3A inhuman liver samples. More specifically there was a strict correlationonly with the expression of CYP3A enzymatic activity (r² 0.993) and notwith other CYP isoenzymes such as CYP1A2 (r² 0.0014), CYP2D6 (r²0.0047), CYP2C9 (r² 0.45) and CYP2C19 (r² 0.0032). The inhibition ofnemorubicin metabolism by human liver microsomes was tested usingantibodies raised against specific cytocrome P-450 isoenzymes. Obtainedresults showed that only the CYP3A4 isoenzyme is responsible for themetabolism of nemorubicin. This finding was further supported by thestudies performed with microsomes obtained from cells tranfected for theoverexpression of different CYP isoenzymes. Only microsomes fromCYP3A4-overexpressing cells were able to metabolize nemorubicin. Theevidence from the above experiments indicates that the CYP3A4 family ofcytocrome P450s is involved in the metabolism of nemorubicin.

The above-obtained results indicate a major role of CYP3A-mediated drugmetabolism in the transformation of nemorubicin. Consequently, being theantitumor activity and host toxicity of nemorubicin affected by thelevel of active/cytotoxic metabolite/s, the CYP3A expression could playa fundamental role in the pharmacological profile of this drug.

Nemorubicin may be therefore considered as an example of an excellentcandidate for an individualized therapy because it is metabolizedprimarily by CYP3A, an enzyme that is known to have interindividualvariability.

There is therefore a need to identify levels of CYP3A in a patient inneed of a treatment with a drug, which is metabolized primarily byCYP3A, so that administration of said drug can be optimized in view ofCYP3A enzymatic profile. Particularly, there is a need to identifylevels of CYP3A in a patient in need of nemorubicin treatment, so thatnemorubicin administration can be optimized in view of CYP3A enzymaticprofile.

The present invention fulfills such a need by providing a method fortreating a patient in need of a treatment with a drug which ismetabolized primarily by CYP3A, especially nemorubicin, which comprisesdetecting CYP3A levels in said patient.

In particular, the present invention is directed to a method foroptimizing therapeutic efficacy of a drug which is metabolized primarilyby CYP3A, especially nemorubicin, in a patient in need thereof, whichcomprises predicting the sensitivity of a patient towards said drugthrough the detection of CYP3A levels in a biological sample of saidpatient and selecting a therapeutically effective amount of said drugbased on the above CYP3A levels.

A further object of the present invention is a method for treating acancer sensitive to a drug which is metabolized primarily by CYP3A,especially nemorubicin, which comprises:

(a) obtaining a biological sample from a patient suffering from saidcancer,

(b) detecting the amount of cytochrome CYP3A in said sample; and

(c) selecting a therapeutically effective amount of said drug, based onthe above cytochrome CYP3A levels.

Another object of the present invention is a method for predictingpatient's sensitivity to a drug, wherein said drug is metabolized byCYP3A, especially nemorubicin, said method comprising determining levelsof CYP3A in said patient and wherein the patient's sensitivity to saiddrug is effected by CYP3A activity.

A kit for detecting the amount of CYP3A in a biological sample for usein a method for treating a cancer sensitive to a drug primarilymetabolized by CYP3A, especially nemorubicin, as described in thepresent specification is also within the scope of the present invention.

For example, according to the patient specific optimal dosing regimenembodiment of the present invention, patients who are candidates fortherapy with a drug primarily metabolized by CYP3A (e.g. nemorubicin)provide, e.g. a biological fluid sample for analysis prior to initiationof the treatment. Suitable, rapid and noninvasive methods and kits suchas, e.g., erytromycin breath test EBT (Rivory et al, Clin Cancer Res.2000), are commercially available for testing CYP3A expression inpatients.

EBT is a putative in vivo probe for drug metabolism by cytocrome P4503A.As an example, specimens of blood may be collected for testing EBT as atool for predicting metabolism of a drug primarily metabolized by CYP3A,e.g. nemorubicin (Rivory et al. Clin. Cancer Res. 2000). Since specificcytochrome P4503A levels determine the tolerance of individual patientsto a particular dose of the above mentioned drug, a math formula can beapplied to calculate a starting dose that minimizes an individualpatient's risk of toxicity and maximizes a patient's probability of atherapeutic responses on the basis of levels of CYP3A found in thebiological samples collected from the patient under examination. Such anindividually adapting starting dose can be greater or smaller than thestarting doses determined empirically in clinical trials that did nottake into account the enzymatic profile of clinical trial participants.

As used herein, “detection” refers to CYP3A level determination inpatients to be treated with nemorubicin.

As used herein, “anticancer therapy” refers to all types of therapiesfor treating cancers or neoplasms or malignant tumors found in mammalscomprising humans, including leukemiae, melanoma, liver, breast, ovary,prostate, stomach, pancreas, lung, kidney, colon and central nervoussystem tumors.

The phrase “therapeutically effective amount” is intended to qualify theamount of nemorubicin, which should be administered to patients, basedon the CYP3A level.

As already said, nemorubicin may be used in anticancer therapy fortreating, e.g. breast, ovary, prostate, lung, colon, kidney, stomach,pancreas, liver, melanoma, leukemiae and central nervous system tumorsin mammals, including humans. In a preferred embodiment, nemorubicin maybe useful for treating a liver cancer, for example a liver cancerprimarily confined to the liver such as, e.g. a hepatocellular carcinomaor a cholangiocarcinoma, or liver metastases.

Nemorubicin can be administered to a patient in any acceptable mannerthat is medically acceptable including orally, parenterally, or withlocoregional therapeutic approaches such as, e.g., implants. Oraladministration includes administering nemorubicin in a suitable oralform such as, e.g., tablets, capsules, lozenges, suspensions, solutions,emulsions, powders, syrups and the like. Parenteral administrationincludes administering nemorubicin by subcutaneous, intravenous orintramuscular injections. Implants include intra artherial implants, forexample an intrahepatic arthery implant.

Injections and implants are preferred administration routes fornemorubicin because they permit precise control of the timing and dosagelevels used for administration.

For example, for treating a patient suffering from a liver cancer asdefined above, intrahepatic administration of nemorubicin may beperformed via the hepatic artery. More precisely, nemorubicin may beadministered to a patient with either a hepatic metastatic cancer, orwith previously untreated primary liver carcinoma, via the hepaticartery directly into the lateral entry of an i.v. line inserted into thebung of an intrahepatic potacath or via a catheter inserted into thehepatic artery.

The actual preferred method of administration of nemorubicin may varyaccording to, inter alia, the particular cancer being treated, theseverity of the disease state being treated, and the particular patientbeing treated.

Pharmaceutically acceptable carriers or excipients to be utilized in thepreparation of a pharmaceutical composition comprising nemorubicin as anactive ingredient are well known to people skilled in the art offormulating compounds in a form of pharmaceutical compositions.

For example, such pharmaceutical compositions may routinely contain,e.g., pharmaceutically acceptable salts, buffering agents, preservativesand/or compatible carriers. As used-herein, “pharmaceutically acceptablecarrier” refers to one or more compatible solid or liquid filler,diluent or encapsulating substances which are suitable foradministration to mammals including humans.

Pharmaceutical compositions suitable for parenteral or intrahepaticadministration are formulated in a sterile form.

The sterile composition thus may be a sterile solution or suspension ina non-toxic parenterally acceptable diluent or solvent.

Pharmaceutical compositions for intrahepatic administration areformulated, for example, in a form, which remains selectively in a livertumor after their injection through the hepatic artery; LIPIODOL™ is asuitable carrier of anticancer agents, which can be used forintrahepatic administration.

The amount of an active ingredient contained in the pharmaceuticalcomposition according to the invention may vary quite widely dependingupon many factors such as e.g. the administration route and the vehicle.

As an example, the pharmaceutical composition of the invention maycontain from 0.1 mg to 100 mg of nemorubicin. In particular, the presentinvention provides a method of treating patients suffering from aprimary or metastatic liver cancer.

In the method of the subject invention, for the administration ofnemorubicin, the course of therapy generally employed is from about 0.1mg/r² to about 1000 mg/m² of body surface area. More preferably, thecourse of therapy employed is from about 1 mg/m² to about 1000 mg/m² ofbody surface area.

1. A method for treating a patient in need of a drug metabolizedprimarily by CYP3A, which comprises detecting CYP3A levels in saidpatient.
 2. The method of claim 1, wherein the drug metabolizedprimarily by CYP3A is nemorubicin.
 3. A method for optimizing thetherapeutic efficacy of a drug metabolized primarily by CYP3A in apatient in need thereof, which comprises predicting the sensitivity ofthe patient towards said drug through the detection of CYP3A levels in abiological sample of said patient and selecting a therapeuticallyeffective amount of said drug based on the above CYP3A levels.
 4. Themethod of claim 3, wherein the drug metabolized primarily by CYP3A isnemorubicin.
 5. A method for treating a cancer sensitive to a drugmetabolized primarily by CYP3A, which comprises: (a) obtaining abiological sample from a patient suffering from said cancer; (b)detecting the amount of CYP3A in said sample; and (c) selecting atherapeutically effective amount of said drug based on the above CYP3Alevels.
 6. The method of claim 5, wherein the drug metabolized primarilyby CYP3A is nemorubicin.
 7. A method for predicting patient'ssensitivity to a drug, wherein said drug is metabolized by CYP3A, saidmethod comprising determining levels of CYP3A in said patient andwherein the patient's sensitivity to said drug is effected by CYP3Aactivity.
 8. The method of claim 7, wherein the drug metabolized byCYP3A is nemorubicin.
 9. A kit for detecting the amount of CYP3A in abiological sample for use in a method for treating a cancer sensitive toa drug metabolized by CYP3A.
 10. The kit of claim 9, wherein the drugmetabolized by CYP3A is nemorubicin.